S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy

Summary p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.

In cells, p62 promotes the assembly and removal of ubiquitinated proteins by forming insoluble cytoplasmic inclusions known as p62 bodies. 21p62 bodies have liquid-like properties formed by polyubiquitin chain-triggered liquid-liquid phase separation, 24,25 which is required for p62 body formation and p62-mediated selective autophagy. 26,279][30][31][32][33] Although the interaction of membrane-associated LC3 with the LIR of p62 is required for p62-mediated selective clearance of ubiquitinated proteins, 22 it remains unclear how autophagosomes efficiently sequester phase-separated p62 droplets.As p62 condensates are unique and important structures in regulating selective autophagy, it is of critical importance to understand molecular mechanisms underlying p62 condensate recruitment to autophagic membranes.
Protein S-acylation is the covalent attachment of a longchain fatty acid to specific cysteine residues within a protein; often, this fatty acid is palmitate or stearate. 34,350][41] In the present study, we report that p62 undergoes reversible S-acylation, which is mediated by the zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and the acyl protein thioesterase 1 (APT1).Mechanistically, S-acylation enhances the affinity of p62 for LC3-positive membranes and promotes autophagic membrane localization of p62 droplets, leading to the production of small LC3-positive p62 droplets, and the subsequent degradation of p62 and ubiquitinated substrates through autophagy.Our results reveal an important molecular mechanism underlying p62 droplet recruitment to the autophagic membrane.

RESULTS p62 is S-acylated in mammalian cells
To measure whether p62 is an acylated protein, we performed an acyl-biotin exchange (ABE) assay, which selectively replaces thioester-linked acyl groups with biotin that can be detected by western blotting probed with streptavidin-horseradish peroxidase (HRP) (Figure S1A). 42We observed the apparent acylation of endogenous p62 in human cervical cancer HeLa cells and normal rat kidney (NRK) cells (Figure 1A).In addition, we also found that exogenously expressed p62 was acylated in breast cancer MCF7 cells (Figures S1B and S1C), consistent with other proteomic studies. 43,44We performed further studies using chemical reporters of protein S-acylation (Figures S1D and  S1E), 45 and we confirmed that p62 is S-acylated through thioester bonds that are cleavable by treatment with hydroxylamine (HAM [NH 2 OH]) (Figure 1B).Specifically, p62 was efficiently labeled by an alkynyl stearic acid (Alk-C18) probe but not significantly labeled by a probe with a chain length of C16 (Alk-C16) (Figure 1B).We then examined p62 S-acylation by ABE assay in cells under autophagy induction (starvation or treatment with the mammalian target of rapamycin [mTOR] inhibitor Torin 1), proteasomal inhibition (treatment with the proteasome inhibitor MG132), or translation inhibition (treatment with the translation inhibitor puromycin).We found that proteasomal inhibition increased the S-acylation of p62 (Figure 1C).Thus, p62 undergoes S-acylation in mammalian cells.

Cys289 and Cys290 of p62 are S-acylated
We next determined the acylation sites of p62.The human p62 protein contains 14 cysteine residues of which six cysteine residues were predicted to be acylated by GPS-and CSS-palm software (Figure S1F). 46,47To verify modification sites experimentally, we individually mutated each predicted cysteine to serine and examined the acylation levels of the mutants by ABE assay.Compared with p62 wild type (WT), the mutants p62 C289S and p62 C290S showed markedly reduced acylation levels, and the double-Cys mutant p62 C289,290S showed almost undetectable acylation (Figures 1D and S1G), suggesting that Cys 289 and Cys 290 are S-acylated.
To further define the region of p62 required for acylation, we constructed fusion motifs consisting of FLAG-GFP and peptides derived from p62, including p62 (285-294), p62 (283-296), and p62 (279-300), and we then examined the acylation levels of these fusions by ABE assay (Figure 1E).We observed that p62 (279-300), but not the double-Cys mutant, was acylated (Figure 1F), suggesting that the p62 (279-300) region is required for p62 S-acylation.Moreover, the acylation sites, Cys 289 and Cys 290 , are conserved among species and are located in an intrinsically disordered region of p62 ranging from 264 to 390 that has been shown to be critical for p62-mediated selective autophagic clearance of ubiquitinated proteins (Figure 1G). 21,48herefore, these results consistently suggest that Cys 289 and Cys 290 are the acylation sites of p62.

S-acyltransferase ZDHHC19 catalyzes p62 S-acylation
Protein S-acylation is catalyzed by a family of ZDHHC domaincontaining S-acyltransferases. 49,502][53] To identify which S-acyltransferases were responsible for p62 S-acylation, each of the 23 hemagglutinin (HA)-tagged ZDHHC enzymes was expressed in cells, and the acylation level of endogenous p62 was subsequently analyzed by ABE assay.We observed an increase in endogenous p62 acylation levels following the expression of ZDHHC4, 5, 7, 19, or 24, with ZDHHC19 being the most efficient (Figures S2A-S2C).ZDHHC19 also showed the clearest colocalization with p62 (Figures 2A, S2D, and S2E).To test the interaction between ZDHHC19 and p62, we performed a coimmunoprecipitation (coIP) assay of HA-ZDHHC19 and FLAG-p62 in HEK293T and NRK cells.We observed a physical interaction between ZDHHC19 and p62 (Figures 2B, 2C, and S2F).Furthermore, we performed TurboID-mediated proximity labeling of ZDHHC19 (Figure 2D) 54 and identified endogenous p62 as a ZDHHC19 interactor in living cells (Figure 2E).To confirm that ZDHHC19 is the endogenous S-acyltransferase mediating p62 modification, we generated ZDHHC19-knockout (KO) HEK293T cells using the CRISPR-Cas9 system (Figures S2G-S2I).We observed that S-acylation of p62 in ZDHHC19-KO cells was substantially reduced compared with that in control cells (Figures 2F and 2G).Taken together, these results suggest that ZDHHC19 catalyzes p62 S-acylation.

S-acylation facilitates the degradation of p62
As an autophagic receptor, p62 is itself degraded along with its cargoes by autophagy.We therefore investigated whether p62 S-acylation is involved in p62 turnover.To achieve this purpose, we expressed plasmids encoding p62 WT or the acylation-deficient mutant p62 C289,290S in WT or p62-KO NRK cells.We found that in both cell lines, a substantial amount of the autophagy marker phosphatidylethanolamine (PE)-modified LC3 (LC3-II) 55  was accumulated upon induction of autophagy with the mTOR inhibitor Torin 1 while degradation of p62 WT was faster than that of p62 C289,290S , which was reversed by the lysosome V-ATPase inhibitor bafilomycin A1 (Baf-A1) (Figures 3A, 3B, and S3A-S3D).
We next examined the effect of increasing p62 acylation on p62 degradation by upregulating ZDHHC19 expression.We observed that the expression of ZDHHC19, but not other ZDHHC enzymes, facilitated p62 degradation under basal condi-tions or upon autophagy induction (Figures S3E and S3F).In contrast, expression of ZDHHC19 C142S , a catalytically inactive ZDHHC19 mutant, did not affect p62 degradation (Figures 3C  and 3D).We further observed a delay in p62 degradation in ZDHHC19-KO cells compared with control cells, which was rescued by re-expressing ZDHHC19 (Figures 3E and 3F).To exclude the possibility that such changes of p62 level were due to lysosomal activity, we evaluated lysosome function upon ZDHHC19 deficiency or overexpression.We found no change in lysosomal pH in ZDHHC19-KO cells and cells transiently expressing ZDHHC19, compared with control cells (Figures S3G and S3H).In addition, ZDHHC19 expression did not change p62 protein levels in autophagy-deficient ATG7-KO HEK293T cells, suggesting that ZDHHC19-mediated p62 degradation is dependent on the autophagy pathway (Figures 3G  and 3H).
S-acylation promotes p62-mediated selective autophagic flux p62 mediates the delivery of ubiquitinated substrates into autophagosomes, and the latter fuse directly with lysosomes for degradation. 56We observed that p62-KO led to accumulation of ubiquitinated proteins, which was fully rescued by re-expressing p62 (Figures 3I and 3J), consistent with previous studies. 57,58owever, cells re-expressing the S-acylation-deficient mutant p62 C289,290S retained the clearance defect for ubiquitinated proteins, suggesting that p62 S-acylation is required for p62mediated selective autophagic clearance of ubiquitinated proteins.In addition, expression of ZDHHC19 facilitated degradation of ubiquitinated proteins, which was blocked by Baf-A1 (Figures 3K, 3L, and S3I), while p62 KO inhibited ZDHHC19induced degradation of ubiquitinated proteins (Figures S3J and  S3K), suggesting that ZDHHC19-mediated degradation of ubiquitinated proteins is dependent on p62.We also observed a delay in the clearance of ubiquitinated proteins in ZDHHC19-KO cells compared with control cells, which was rescued by re-expressing ZDHHC19 (Figures 3E and S3L).
To further investigate the effect of p62 S-acylation on selective autophagic flux, we treated p62-KO NRK cells stably expressing p62 WT or p62 C289,290S with Baf-A1 and then assessed the accumulation of ubiquitinated proteins.We observed that lysosomal inhibition caused greater accumulation of ubiquitinated proteins in p62 WT cells than in p62 C289,290S cells (Figures S3M  and S3N).Furthermore, lysosomal inhibition led to the accumulation of poly-ubiquitinated proteins upon ZDHHC19 expression (Figures 3M and 3N), suggesting that ZDHHC19-mediated p62 S-acylation promotes p62-mediated selective autophagic flux, thereby facilitating the entry of ubiquitinated substrates into autophagosomes.
To determine the physiological function of p62 S-acylation, we compared the viability of these cell lines.We observed that p62 C289,290S NRK cells and ZDHHC19-KO HEK293T cells showed reduced viability compared with p62 WT NRK cells and HEK293T cells, respectively, upon proteasomal inhibition (Figures S3O-S3Q), suggesting an important physiological role for p62 S-acylation upon induction of proteotoxic stress.
Taken together, these results suggest that ZDHHC19-catalyzed p62 S-acylation facilitates p62-mediated selective autophagy, which indicates an important role for p62 S-acylation in cell survival under stress.
S-acylation promotes the formation of small p62 droplets Next, we investigated why p62 S-acylation facilitates degradation of p62 and ubiquitinated proteins through autophagy.p62 tends to form cytoplasmic aggregates with ubiquitinated proteins and is often observed in discrete punctate structures known as cellular p62 bodies in cells. 24,25We therefore tested the effect of S-acylation on the formation of p62 puncta.Surprisingly, we observed, under similar levels of GFP-p62 WT and GFP-p62 C289,290S , a significant accumulation of p62 puncta with a diameter greater than 1 mm (large p62 puncta) 59 in GFP-p62 C289,290S -expressing cells compared with GFP-p62 WT-expressing cells (Figures 4A-4C), suggesting that deficiency in p62 S-acylation results in the formation of large p62 puncta.This finding was confirmed in ZDHHC19-KO cells, where we also observed large p62 puncta (Figures 4D and 4E).Also, the expression of ZDHHC19, but not the catalytically inactive mutant ZDHHC19 C142S , enabled the formation of smaller p62 puncta compared with that of the control (Figures 4F and 4G).Furthermore, transient expression of ZDHHC19 did not distribute p62 bodies into small puncta in GFP-p62 C289,290S -expressing NRK cells, suggesting that the formation of small p62 droplets   induced by ZDHHC19 is dependent on S-acylation at Cys289 and Cys290 of p62 (Figures 4H and 4I).
Previous studies have suggested that binding affinity of p62 and ubiquitinated proteins, as well as self-oligomerization of p62, determines the size distribution of p62 droplets. 6,28,30,60e observed that both GFP-p62 puncta and large GFP-p62 C289,290S puncta co-localized with ubiquitin (Figure S4A).In addition, p62 WT and p62 C289,290S co-precipitated equivalent amounts of ubiquitinated proteins (Figure S4B), consistent with in vitro pull-down data (Figure S4C).Using non-reduced SDS-PAGE, we observed the same amount of p62 monomer and oligomer in p62 WT-or p62 C289,290S -expressing cells (Figure S4D).We further found that the overexpression of ZDHHC19 did not affect the distribution of p62 monomer and oligomer (Figure S4E).Moreover, coIP analysis between p62 with different tags showed that S-acylation did not affect PB1 domain-dependent p62-p62 interaction (Figures S4F-S4I).These results suggest that p62 Sacylation has no effect on p62-ubiquitin binding and its oligomerization.
Therefore, p62 S-acylation mediated by ZDHHC19 promotes the formation of small p62 droplets, and deficiency in p62 S-acylation leads to the formation of large p62 droplets, which is not due to changes in p62-ubiquitin binding or oligomerization.

S-acylation regulates autophagic membrane localization of p62 droplets
We next investigated why p62 S-acylation promotes the formation of small p62 droplets.p62 bodies have viscous liquid-like properties. 24,61p62 puncta are observed both as membranefree protein aggregates or as being engulfed by autophagosomes or autolysosomes. 21Studies have suggested that LC3positive membranes associate with p62 droplets and deform p62 droplets through a wetting process, a phenomenon in which liquid-like properties of droplets allow for droplet deformation into spherical caps when a droplet contacts a surface. 60,627][38] We therefore investigated whether p62 S-acylation regulates autophagic membrane localization of p62 droplets.In GFP-p62 WT-expressing cells, p62 puncta showed co-localization with the autophagosome marker LC3 upon autophagy induction and became small p62 puncta engulfed by autophagosomes (Figures 5A and 5B), in agreement with previous studies. 21In comparison, in GFP-p62 C289,290S -expressing cells, fewer p62 puncta were recruited to autophagosomes upon autophagy induction.To exclude the possibility that such changes of p62 co-localization with LC3 were due to impairment of p62-LC3 interaction, we performed a coIP analysis of p62 WT or p62 C289,290S and LC3.We observed that HA-tagged LC3 co-immunoprecipitated the same amount of FLAG-p62 WT and FLAG-p62 C289,290S , suggesting that S-acylation has no effect on p62-LC3 interaction (Figure S5A).
We next performed co-localization analysis of p62 puncta and LC3-positive membranes in the presence or absence of ZDHHC19.The expression of ZDHHC19, but not ZDHHC19 C142S , induced an increase in LC3-positive endogenous p62 puncta in HEK293 cells stably expressing mCherry-LC3 (Figures 5C and 5D).In ZDHHC19-KO cells, endogenous p62 formed abundant large puncta that were LC3-negative upon autophagy induction (Figures 5E and 5F).In addition, we observed small LC3-positive p62 puncta in GFP-p62 WT-expressing cells upon HA-ZDHHC19 expression, while it was difficult to establish LC3 localization owing to the large p62 puncta in GFP-p62 C289,290S -expressing cells (Figure 5G).The large p62 WT puncta adhered to LC3 dots, whereas it was difficult to observe apparent LC3 dots on large p62 C289,290S puncta (Figure S5B; Videos S1 and S2).Consistently, we found that, compared with GFP-p62 WT, more GFP-p62 C289,290S accumulated in the detergent-insoluble fraction (Figure S5C).Similarly, ZDHHC19-KO led to p62 accumulation in the detergent-insoluble fraction (Figure S5D).We then tested whether S-acylation regulates the liquid-like state of p62 by fluorescence recovery after photobleaching (FRAP), commonly used to measure condensate fluidity. 63We observed that GFP-p62 had a faster recovery time than GFP-p62 C289,290S (Figure 5H), suggesting that S-acylation could regulate physicochemical behavior of p62-liquid droplets.Based on above results, we conclude that S-acylation mediates the association of p62 bodies with autophagic membranes, leading to formation of smaller LC3-positive p62 condensates.
To better understand the effect of S-acylation on autophagic membrane localization of p62 droplets, we determined where ZDHHC19-mediated p62 S-acylation occurs.ZDHHC19 itself was strongly co-localized with endoplasmic reticulum (ER) membranes as well as autophagic membranes (Figures S5E-S5I).Furthermore, we observed that autophagy inhibition by FIP200 deletion caused an accumulation of S-acylated p62 in HeLa and mouse embryonic fibroblast (MEF) cells (Figures S5J-S5M), indicating that ER-localized ZDHHC19 mediates p62 S-acylation in the ER, and then follows p62 to autophagic membranes.
These results suggest that ZDHHC19-mediated S-acylation enhances autophagic membrane localization of p62 droplets, facilitating efficient degradation of their substrates.

S-acylation enhances the affinity of p62 for LC3-positive membranes in vitro
To directly test whether S-acylation promotes the interaction of p62 with LC3-positive membranes, we performed in vitro assays using acylated p62 protein and membrane mimics.Using a bioconjugation strategy, a palmitate with a maleimide moiety was reacted with purified recombinant protein to produce p62 protein-palmitate conjugates to mimic acylated proteins (Figure S6A), which is widely used to study the function of lipidated (G) Representative images of p62 puncta and autophagosome in GFP-p62 WT or GFP-p62 C289,290S cells upon ZDHHC19 expression.p62-KO NRK cells stably expressing GFP-p62 WT or GFP-p62 C289,290S (green) were transfected with HA-ZDHHC19 and immunostained with anti-LC3A/B antibody (red) and anti-HA antibody (purple).Scale bars, 10 mm.(H) Fluorescence recovery after photobleaching (FRAP) assays of GFP-p62 WT and GFP-p62 C289,290S puncta.Data from four independent experiments were analyzed.Scale bars, 1 mm.See also Figure S5.
We first investigated whether S-acylation promotes the interaction of p62 with membranes.Giant unilamellar vesicles (GUVs) are artificial membrane-mimetic systems used for the study of membrane-related phenomena.The system is characterized by its unilamellar lipid bilayer and micrometer-wide globular shape, allowing for easy observation of membrane interactions by microscopy.We observed that FITC-labeled p62 (279-300)-palmitate peptide was recruited to functionalized GUV membranes containing a red fluorescence rhodamine dye within 60 min, whereas the unmodified p62 peptide did not bind to GUVs (Figure S6F; Video S3).Similarly, acylated GFPtagged p62 (155-440), but not the unmodified protein, was recruited to GUVs (Figure 6A; Video S4).In addition, we evaluated the affinity of acylated p62 protein to total cellular membranes in vitro (Figure 6B).We observed that while unmodified GFP/ mCherry-p62 proteins targeted the membrane inefficiently, most acylated p62 proteins were recruited to cellular membranes, with the protein level in the supernatant decreasing accordingly (Figures 6C and 6D).These results suggest that acylation enhances the affinity of p62 protein to artificial membranes and total cellular membranes.In addition, we found that the association of p62 to isolated cellular membranes was faster than to artificial GUVs, indicating that other membraneassociated proteins or membrane composition has an impact on membrane association of p62.
We next evaluated the interaction between acylated p62 and LC3-positive or LC3-negative membranes.To this purpose, we treated total cellular membranes with the recombinant bacterial enzyme RavZ that can irreversibly delipidate membrane-associated LC3-II on autophagic membranes and release LC3 protein from membranes (Figures 6E and S6D). 66,67We observed that more acylated p62 protein accumulated to total cellular membranes than that of RavZ-treated membranes (Figure 6F).Moreover, total cellular membranes were isolated from HEK293T WT cells or ATG7-KO HEK293T cells (Figure 6E).There was no LC3-II protein in the cell membranes from ATG7-KO HEK293T cells, because E1-like ATG7 deficiency prevents production of LC3-II in these cells.We observed that acylated p62 accumulated to the total cellular membranes from HEK293T WT cells but not to membranes from ATG7-KO cells (Figure 6G).These results suggest that acylated p62 has higher affinity for LC3-positive membranes than that for LC3-negative membranes.
To further confirm the binding of S-acylated p62 and membranes, we reconstituted S-acylated p62 in cellular membranes in vitro (Figure 6H).To this purpose, we purified S-acylated p62 and non-acylated p62 from HEK293T transiently expressing FLAG-p62 WT and FLAG-p62 C289,290S , respectively, and isolated total cellular membrane from p62-KO NRK cells.On incubation with total cellular membrane, more acylated p62 proteins were accumulated than non-acylated p62 (Figure 6I).
Therefore, these in vitro results are consistent with an increased affinity of acylated p62 to LC3-positive membranes.

APT1 deacylates p62
Deacylation, the reversal of S-acylation, is mediated by acyl protein thioesterases (APTs). 68Previous studies have identified several mammalian deacylases, including APTs (APT1 and APT2), palmitoyl protein thioesterases 1 and 2 (PPT1 and PPT2), and the diverse a/b-hydrolase fold domain (ABHD) family of proteins. 69,70To identify the enzyme that catalyzes the deacylation of p62, we expressed five known APTs in MCF7 cells and detected the S-acylation level of endogenous p62 by ABE assay.We found that expression of APT1, but not other APTs, significantly decreased endogenous p62 S-acylation (Figure 7A).
Given that p62 S-acylation facilitates p62 degradation, we then investigated the role of APT1-mediated p62 deacylation in the regulation of p62-mediated selective autophagy.We first checked p62 protein levels upon APT1 expression or in APT1-KO cells (Figures S7A-S7C).We observed that APT1 expression inhibited p62 turnover and degradation of ubiquitinated proteins (Figures 7B-7D).It is noted that Baf-A1 treatment increased levels of p62, but not ubiquitinated proteins, in cells expressing the APT1 S119A mutant, suggesting that APT1 could affect ubiquitinated protein degradation in a manner partially independent of p62-mediated autophagy.Compared with control WT cells, p62 protein level was decreased in APT1-KO cells, which was recovered by re-expressing APT1 but not catalytically inactive mutant APT1 S119A (Figures 7E and 7F).We then determined the effect of (C) Immunoblot analysis of mCherry-p62 and mCherry-p62-palm proteins co-precipitated with total cellular membrane in vitro as in (B).The levels of Na + /K + ATPase and GADPH were detected as internal references for total cellular membrane and cytoplasm, respectively.(D) Immunoblot analysis of GFP-p62 and GFP-p62-palm proteins co-precipitated with total cellular membrane in vitro as in (B).(E) Schematic diagram of GFP-p62-palm co-precipitated with LC3-positive or LC3-negative membranes.(F) Immunoblot analysis of GFP-p62-palm co-precipitated with LC3-rich and RavZ-treated membrane in vitro as in (E).(G) Immunoblot analysis of GFP-p62-palm co-precipitated with LC3-positive and LC3-negative membrane from in vitro ATG7-KO HEK293T cells as in (E).(H) Schematic diagram of reconstitution of S-acylated p62 in total cellular membrane in vitro.S-acylated p62 and non-acylated p62 proteins were purified from p62-KO NRK cells transiently expressing FLAG-p62 WT and FLAG-p62 C289,290S , respectively.The total cellular membranes were prepared from p62-KO NRK cells and further incubated with the purified p62 proteins.(I) Immunoblot analysis of mammalian cell-expressed FLAG-p62 WT and FLAG-p62 C289,290S proteins co-precipitated with total cellular membrane in vitro as in (H).See also Figure S6.
Taken together, we conclude that APT1 deacylates p62 and regulates its function by suppressing p62-mediated selective autophagy.

DISCUSSION
Previous studies have shown that p62-mediated selective autophagy is regulated by several posttranslational modifications of p62, such as phosphorylation, acetylation, and ubiquitination. 6,28,30In this study, we revealed a posttranslational modification of p62 with long-chain fatty acids, known as S-acylation.We have demonstrated that S-acylation of p62 enhances the affinity of p62 for LC3-positive membranes and promotes p62 droplet recruitment to autophagosomes.S-acylation of p62 is dynamically regulated by S-acyltransferase ZDHHC19 and APT1.These data highlight the importance of S-acylation in regulating p62-mediated selective autophagic clearance of ubiquitinated proteins in response to proteotoxic stress.Specifically, p62 S-acylation facilitates entry of p62 droplets into autophagosomes for degradation, hence enhancing p62-mediated selective autophagic flux.S-acylation promotes autophagic membrane localization of p62 droplets and the formation of small LC3-positive p62 droplets.Our study shows the functional significance of the S-acylation-deacylation cycle in regulating p62 droplet recruitment to the autophagic membrane, advancing our understanding of the molecular mechanism and regulatory network of p62-mediated selective autophagy.
Autophagy is a highly dynamic intracellular trafficking process and involves various membrane-related events that are tightly regulated by autophagy-related proteins. 71Recently, proteomic studies of S-acylated proteins showed that many autophagyrelated proteins are putatively acylated. 72Our work will open up a field of investigation on the mechanism of autophagyrelated protein interactions with autophagic membranes.
S-acylation regulates p62 droplet association with autophagic membrane S-acylation has previously been shown to increase the hydrophobicity of proteins and to contribute to their membrane association.Indeed, our results suggest that S-acylation enhances the association of p62 to membrane mimics such as artificial membranes and isolated cellular membranes.We were also able to reconstitute S-acylated p62 in cellular membranes in vitro, enabling us to show that acylated p62 associated more with LC3-positive membranes than with LC3-negative membranes, which suggests that membrane-associated LC3 assists the interaction between S-acylated p62 and membranes in vitro.
Our results also show that deficiency or a decrease in p62 Sacylation results in the formation of large LC3-negative p62 puncta in cells, indicating that S-acylation plays an important role in regulating p62 droplet engulfment by the autophagic membrane.It is known that p62 binds directly to LC3 via its LIR motif to facilitate the degradation of ubiquitinated protein aggregates by autophagy. 22It seems that in response to ubiquitin proteotoxic stress, p62 is S-acylated by the ER membraneassociated ZDHHC19; subsequently, p62 links to the autophagic membrane by its lipid and LIR motif.S-acylation-mediated association of p62 droplets with the autophagic membrane coupled with the p62-LC3 interaction could yield enhanced affinity for p62 droplets and the autophagic membrane, thereby leading to the efficient recruitment of p62 droplets by the autophagic membrane.This is an example of S-acylation in regulating biomolecular condensate association with membrane.

S-acylation facilitates deformation of p62 droplets by autophagic membrane
Intracellular wetting mediates contacts between p62 droplets and autophagosomal membranes, leading to formation of multiple small LC3 puncta corresponding to autophagosomal membranes on the surface of these droplets. 60,62,73Supporting this notion, we observed small LC3-positive puncta upon autophagy induction.Deficiency or decreasing p62 S-acylation results in the production of large p62 puncta, whereas increasing p62 S-acylation promotes the formation of small LC3-positive p62 droplets.These findings are consistent with a model whereby ZDHHC19mediated p62 S-acylation promotes autophagic membrane localization of p62 droplets and enhances the interaction of p62 droplets with autophagic membranes.We propose that Sacylation enhances autophagic membrane association of p62 droplets and hence promotes wetting of p62 droplets with the autophagic membrane, thereby contributing to the production of small LC3-positive p62 droplets and efficient degradation of their substrates.

Increasing p62 acylation enhances p62-mediated selective autophagic flux
There is mounting evidence that dysregulation of selective autophagic clearance of protein aggregates is linked to a variety of pathological processes, including neurodegenerative diseases. 10,11,74,757][78] Most chemical inducers targeting common autophagy pathways activate selective autophagy; however, these chemical inducers also trigger basal autophagy activity, thereby leading to unexpected side effects. 79,80Targeting specific components of selective autophagy machineries would be a promising alternative for the development of new autophagy inducers. 81Our results indicate that increasing p62 acylation by upregulation of ZDHHC19 or genetic KO of APT1 accelerates selective autophagy by promoting the entry of ubiquitinated substrates into autophagy but has no effect on the level of LC3-II (Figures 7H and S7A), suggesting that increasing p62 acylation shows high selectivity for autophagic clearance of ubiquitinated proteins.Therefore, our study provides a strategy to specifically enhance selective autophagic clearance of protein aggregates.A recent report showed that APT1 inhibitor restores Huntington's disease (HD) by increasing brain palmitoylation, 82 which is line with our notion that APT1 inhibition could provide a potential strategy against neurodegenerative diseases.

Limitations of the study
Our study provides strong evidence that S-acylation enhances the association of p62 to LC3-positive membranes and further promotes p62 droplet recruitment into autophagosomes.p62 is Sacylated by the ER membrane-associated ZDHHC19, but exactly how S-acylated p62 droplets arrive at autophagosomes remains to be explored.In addition, in the absence of p62, ER membrane-associated ZDHHC19 was still strongly co-localized with autophagic membranes, and ZDHHC19 and the catalytically inactive mutant ZDHHC19 C142S were still degraded by autophagy.Although there was no apparent change in LC3-II level upon expression or deficiency of ZDHHC19, future research should explore other substrates and non-catalytic functions of ZDHHC19 in autophagy.Finally, while this research highlights the significance of p62 S-acylation in cell survival under stress, future studies should investigate the functional significance in animal models.
(Sun Yat-Sen University) for FIP200-KO HeLa cells; Prof. Yueguang Rong (Huazhong University of Science and Technology) for FIP200-KO MEF cells; Masaki Fukata (National Institute for Physiological Sciences) for the expression vectors of the ZDHHC proteins; and Jieqiong Tan (Central South University) for the plasmid encoding human p62.We are also thankful for the technical support by the Analytical and Testing Center of Chongqing University.This study was supported by the National Natural Science Foundation of China (NSFC) (91854101, 32170185, and 31801166), the Natural Science Foundation of Chongqing, China (CSTB2022NSCQ-MSX0463), and the Venture and Inno-

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Aimin Yang (aimin.yang@cqu.edu.cn).

Materials availability
Plasmids and cell lines generated in this study will be available upon request.To delete ZDHHC19 in HEK293T cells, two guide RNAs (gRNAs) against the 3rd exon in the human ZDHHC19 genome were designed, using an online tool (http://crispor.tefor.net/).The forward gRNA sequence (ACGGGGCCTTCCGCCTGCAA) and the reverse gRNA sequence (CACACCACGTGCACCGTCAA) are against the upstream and downstream of exon 3, respectively.The doublestranded oligos for each gRNA were synthesized by Shanghai BiOligo Biotechnology and cloned into the pX458 vector (48138, Addgene).The plasmids were co-transfected into HEK293T cells by LipofectamineÔ 3000 (Thermo Fisher, L3000015).Single GFP-fluorescence cell was sorted by fluorescence-activated cell sorting (FACS) (BD Biosciences) to pick single-cell clones.KO cells were confirmed by genotyping and sequencing PCR-amplified genomic DNA.The pair of primers: forward-5'-CTTGTCCCCA CCCTTCCTTG-3' and reverse-5'-GACAGGTGCAGGTGGAGAAC-3' were used for genotyping.Genomic DNA was extracted using a universal DNA purification kit (TIANGEN, 4992197).Sequencing results showed that the design for CRISPR/Cas9 gene targeting was successful, and 56 bp in the ZDHHC19 genome was missing, causing premature termination of the protein.

Data and code availability
To delete APT1 in HEK293T cells, two guide RNAs (gRNAs) against the 7th exon in the human APT1 genome were designed, using an online tool (http://crispor.tefor.net/).The forward gRNA sequence (GTGTCACTGCACTCAGTTGC) and the reverse gRNA sequence (CAGTTTCTGCTGTGTGGTAA) are against the upstream and downstream of exon 7 respectively.The double-stranded oligos for each gRNA were synthesized by Shanghai BiOligo Biotechnology and cloned into the pX458 vector (48138, Addgene).The plasmids were co-transfected into HEK293T cells by LipofectamineÔ 3000 (Thermo Fisher, L3000015).Single GFP-fluorescence cell was sorted by fluorescence-activated cell sorting (FACS) (BD Biosciences) to pick single-cell clones.KO cells were confirmed by genotyping and sequencing PCR-amplified genomic DNA.The pair of primers: forward-5'-TGGGCAAGCATG TGTGTTC-3' and reverse-5'-ACCTTCTGCTCAGTTTTGCT-3' were used for genotyping.Genomic DNA was extracted using a universal DNA purification kit (TIANGEN, 4992197).Sequencing results showed that the design for CRISPR/Cas9 gene targeting was successful, and 38 bp in the APT1 genome was missing, causing premature termination of the protein.

Acyl-biotin exchange (ABE) assay
The ABE assay was performed as previously described with minor modifications. 36,83In brief, cells transfected with Flag-p62 were harvested 48 h after transfection and washed with cold PBS.Prior to cell lysis, 50 mM N-ethylmaleimide (NEM; Sangon Biotech, A600450) dissolved in EtOH was freshly added to the Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 1% NP-40, 10% glycerol) with inhibitor cocktail.Cells were then suspended in NEM-containing Lysis Buffer for 1.5 h at 4 C and centrifugated at 15,000 rpm for 30 min at 4 C.The supernatants were incubated with both anti-Flag antibody (GenScript, A00187) and protein A/G magnetic beads (MedChemExpress, HY-K0202) at 4 C overnight.For S-acylation detection of endogenous p62, anti-p62 antibody (MBL, PM045) was used to enrich endogenous p62.After incubation, the beads were washed five times with Lysis Buffer of pH 7.5 and then three times with Lysis Buffer of pH 7.2.For the hydroxylamine (HAM; MACKLIN, H828371) treatment, beads were equivalently divided into two tubes (+HAM and -HAM).Then, freshly prepared HAM-containing Lysis Buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 1 mM MgCl 2 , 1% NP-40, 10% glycerol, 0.75 M HAM and inhibitor cocktail) was added to the +HAM tube and incubated at room temperature for 1 h.As a control, the Lysis Buffer of pH 7.2 was added to the -HAM tube.The beads were then washed four times with Lysis Buffer of pH 7.2 and three times with Lysis Buffer of pH 6.2.Subsequently, beads were treated with 5 mM biotin-BMCC (Sangon Biotech, C1002225) in Lysis Buffer of pH 6.2 freshly prepared at 4 C for 1 h.The immunoprecipitated samples were analyzed by Western blotting using HRP-conjugated streptavidin (Beyotime, A0303) and anti-Flag antibody (GenScript, A00187).
LC-MS of HAM-mediated hydrolysate MCF7 cells were harvested and incubated with 50 mM NEM.The supernatants were incubated with both anti-p62 antibody (MBL, PM045) and protein A/G magnetic beads (MedChemExpress, HY-K0202) at 4 C for overnight.After incubation, the beads were washed five times with Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 1% NP-40, 10% glycerol, and inhibitor cocktail) and then three times with Lysis Buffer of pH 7.2.Then, freshly prepared HAM-containing Lysis Buffer of pH 7.2 was added and incubated at room temperature for 1 h.As a control, Lysis Buffer of pH 7.2 with inhibitor cocktail was added.After HAM treatment, the supernatant was collected and subjected to Q Exactive Plus Orbitrap LC-MS (Thermo Fisher).The sample was separated using C18 (3 mm, 75 nm 3 15 cm) (Thermo Fisher) at a flow rate of 600 nL/min.The gradient was set as follows: 3% B (0.1% formic acid in ACN)/97% A (0.1% formic acid in H 2 O) to 8% B for 5 min, 8% B to 20% B for 38 min, 20% B to 30% B for 8 min, 30% B to 90% B for 2 min and 90% B for 7 min.The data were collected in the negative ion and positive ion modes, and stearic and palmitic acids were analyzed in the negative ion mode.

Proximity labeling
Proximity labeling was performed as previously described with minor modifications. 54,85HEK293T cells were grown in DMEM (10% FBS) at 37 C under 5% CO 2 and transfected with the ZDHHC19-TurboID plasmid by LipofectamineÔ 3000 (Thermo Fisher, L3000015).After 24 h, the cells were labeled by 500 mM D-Biotin (Solarbio, D8150) for 40 min at 37 C with fresh DMEM.The cells were then washed four times with cold PBS and collected with cold PBS at 800 g for 5 min.The cell lysates were extracted with RIPA Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate and 1% Triton X-100) for 30 min with rotation at 4 C and centrifuged at 12,000 rpm for 10 min at 4 C. Next, proteins were precipitated by adding CH 3 OH:CHCl 3 :H 2 O at a ratio of 4:1:3, and washed three times with ice-cold CH 3 OH.After air-drying, 2% SDS/PBS buffer was added for protein redissolving at room temperature for 1 h, followed by diluting with PBS to 0.1% SDS/PBS.Then, streptavidin-coated magnetic beads (MedChemExpress, HY-K0208) were incubated with the protein sample for 2 h with rotation at room temperature.The beads were washed once with 0.2% SDS/PBS buffer, three times with PBS buffer and once with ddH 2 O, followed by transferring to a new 1.5 mL tube.23 SDS Sample Buffer with 2 mM D-Biotin and 20 mM DTT was added to the beads for 95 C boiling for 10 min to elute purified proteins for Western blotting analysis.

Biochemical fractionation
Protein aggregates were determined by fractioning cell lysates in Triton X-100 soluble and insoluble fractions as previously described. 86Briefly, cells were rinsed with cold PBS, lysed in PBS containing 2% Triton X-100 supplemented with inhibitor cocktail, and incubated on ice for 1 h.The lysates were centrifuged at 15000 rpm for 40 min at 4 C.The supernatant was recovered as the soluble fraction.Pellets were rinsed with PBS three times and then resuspended in 1% SDS supplemented with inhibitor cocktail and sonicated to obtain the insoluble fraction.Protein was quantified by an Enhanced BCA protein assay kit (Beyotime, P0010) and then determined by Western blotting.

Immunoprecipitation analysis
Cells transfected with HA-ZDHHC19 and Flag-p62, GFP-p62 and Flag-p62, or Flag-p62 and HA-LC3 were lysed by Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40) with inhibitor cocktail.The supernatants were incubated with both antibody and protein A/G magnetic beads (MedChemExpress, HY-K0202) at 4 C for overnight.Subsequently, the beads were washed with cold Lysis Buffer five times.The immunoprecipitated samples were quantified by an Enhanced BCA protein assay kit (Beyotime, P0010) and analyzed by Western blotting using anti-Flag and anti-HA antibodies.
For immunoprecipitation analysis of protein-palmitate conjugate and ubiquitin, HEK293T cells were lysed by Lysis Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40) with inhibitor cocktail.The supernatants were harvested by centrifugation at 15,000 rpm, 20 min, 4 C and were mixed with 20 ng/mL GFP-p62 protein or palmitate-maleimide conjugated GFP-p62 protein.The mixture was then incubated with both anti-GFP antibody (ZENBIO, 300943) and protein A/G magnetic beads (MedChemExpress, HY-K0202) at 4 C for overnight.Subsequently, the beads were washed with cold Lysis Buffer for five times and eluted with SDS Sample Buffer.Ubiquitin and p62 were analyzed by western blotting using anti-Ub antibody (Santa Cruz, sc-8017) and anti-GFP antibody (ZENBIO, 300943), respectively.

Protein expression and purification
Plasmid containing p62 or RavZ was transformed into E. coli BL21 Rosetta (DE3) cells.Protein expression was induced with 0.2-0.4mM IPTG and carried out at 18 C overnight.Purification was performed with the A ¨KTAÔ prime plus chromatography purification system (GE Healthcare Life Sciences).The cells were collected and re-suspended in Lysis Buffer (40 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF) for sonication.All of the proteins used in this study contain tags (GFP, mCherry or MBP) with an extra N-terminal His6 tag.The proteins were purified by Ni-NTA affinity purification using HisTrap HP column (GE Healthcare Life Sciences) eluted with a gradient of 4-70% of 500 mM imidazole.Purified proteins were collected and concentrated by Centrifugal Filtration (30 K NMWL) (Merck Millipore, UFC803096).The concentration of target protein was measured by SDS-PAGE using BSA standard samples.

Peptide synthesis and purification
All peptides were synthesized based on manual Fmoc-SPPS chemistry.Briefly, Rink Amide-MBHA resins (MACKLIN, R916084) with a loading capacity of 0.36 mmol/g were first swelled by DCM.For each coupling procedure, 4-fold excess of protected amino acid, HBTU, HOBt, and DIPEA (with a ratio of 1:1:1:2) in DMF was added to the resin for 90 minutes with shaking at room temperature.The deprotection reaction of the Fmoc group was performed in 20% piperidine in DMF (v/v) after the resins were washed with DMF 5 times.The procedure for the coupling of fluorescein 5-isothiocyanate (FITC; GuoPing Pharmaceutical, GP010852-1) was performed with 3-fold excess of 5-FITC and DIPEA (with a ratio of 1: 2) in DMF overnight.After completion of the synthesis, peptides were released from the resin by treatment with TFA/H 2 O/TIS (95/2.5/2.5) for 2 h at room temperature.The crude peptides were precipitated by the addition of cold diethyl ether (Et 2 O) and centrifugation.
For purification, the crude peptides were injected to RP-HPLC (Hitachi, Japan) equipped with a C18 column (Bondysil C18, 5 mm, 250 mm 3 5.6 mm).0.1% TFA in ACN (v/v) and 0.1% TFA in H 2 O (v/v) were used as the mobile phase A and B, respectively.For all the analytical HPLC trials, the total flow rate was set to be 1 mL/min, and the A concentration raised from 10 % to 100 % over 21 min following a linear gradient.For the purification of peptides in a larger scale by semi-prep HPLC columns (Bondysil C18, 5 mm, 250 mm 3 10 mm), the total flow rate was set to be 3 mL/min and the A concentration raised from 10% to 100% over 21 min following a linear gradient.The peptide peaks were collected, lyophilized, and confirmed by MALDI-TOF mass spectrometry analysis (SHIMADZU Corporation, Japan).

Synthesis of palmitate-maleimide
Scheme 1. Synthesis route of palmitate-maleimide Palmitate-maleimide was prepared according to the previous reported literature (Scheme 1). 87To a 100 mL of round-bottom flask was added triphenylphosphine (PPh 3 ; 1.35 g, 5.2 mmol) and 50 mL of anhydrous tetrahydrofuran (THF).The resulting clear solution was cooled to -78 C. Diethyl azodicarboxylate (DEAD; 0.82 mL, 5.2 mmol) was added over a period of 2-3 min.The yellow mixture was then stirred during 5 min, after which hexadecanol (1.33 g, 5.7 mmol) was added over 1 min and stirred for 5 min.Then neopentyl alcohol (t-BuCH 2 OH; 0.25 g, 2.8 mmol) and maleimide (0.50 g, 5.2 mmol) were added.The resulting suspension was allowed to remain at -78 C for 5 min during which time most of the maleimide dissolved.The cooling bath was then removed, and the mixture was stirred overnight at room temperature.After completion of the reaction, the clear solution was evaporated and the resulting solid was purified by a column chromatography. 1 H NMR (600 MHz, CDCl 3 ) d 6.67 (s, 2H), 3.5 (t, J = 7.2 Hz, 2H), 1.56 (m, 2H), 1.25 (s, 26H), 0.87 (t, J = 6.7 Hz, 3H).

GUV assay
GUV assay was performed as previously described with minor modifications. 881% agarose was used to coat one glass slide for overnight, while the other glass slide was coated by 200 mL streptavidin solution (7 mg streptavidin dissolved in 200 mL ddH 2 O) and dry for overnight.For preparation of GUVs, 40 mL of chloroform was added to 60 mL lipids solution (15 mL 10 mM DPPC, 30 mL 10 mM cholesterol, 12.5 mL 10 mM Biotin-PE, 1 mL 10 mM rhodamine-PE and 1.5 mL 10 mM POPS).The lipids solution was then transferred to the agarose-coated glass slide by glass syringe (HEMILTON) and dry with Argon gas for 3 h.Then, 200 mL PBS was gently added to agarose glass for 10 min to suspend the GUVs, which were subsequently added to the streptavidin-coated glass slide.After 20 min incubation, GUVs were anchored to glass slides through biotin-streptavidin interaction.p62 protein or peptide was injected into the chamber.The fluorescence of GUVs was observed continuously for 60 min using a laser scanning confocal microscope (Olympus IX2).For each representative images, three different experiments were performed to ensure the reproducibility.

Preparation of membrane fraction
To prepare total cellular membrane fraction preparation, HEK293T or p62-KO NRK were scraped and resuspended in PBS.Then cells were frozen by liquid nitrogen and thawed at 37 C for five cycles.The cell nucleus was removed by centrifugation at 1,000 g, 5 min, 4 C. Membrane fraction was achieved by centrifugation at 15,000 rpm, 20 min, 4 C.
To prepare total LC3-positive or LC3-negative cellular membrane, the HEK293T or ATG7-KO HEK293T cells were treated with 1 mM Torin 1 (MedChemExpress, HY-13003) and 1 mM Baf-A1 (Selleck, S1413) for 4 h.The cells were scraped and resuspended in PBS.Then cells were frozen by liquid nitrogen and thawed at 37 C for five cycles.The cell nucleus was removed by centrifugation at 1,000 g, 5 min, 4 C. Membrane fraction was achieved by centrifugation at 15,000 rpm, 20 min, 4 C and washed with PBS.
Alternatively, to prepare total LC3-positive or LC3-negative cellular membrane, the GFP-LC3-expressing MCF7 cells were treated with 1 mM Torin 1 (MedChemExpress, HY-13003) 1 mM Baf-A1 (Selleck, S1413) for 4 h.The cells were scraped and resuspended in PBS.Then cells were frozen by liquid nitrogen and thawed at 37 C for five cycles.The cell nucleus was removed by centrifugation at 1,000 g, 5 min, 4 C.The LC3-positive membrane was obtained by centrifugation at 15,000 rpm, 20 min, 4 C and washed with PBS.To prepare LC3-negative membrane, the LC3-positive membrane was resuspended in Reaction Buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1mM DTT) and incubated with 50 ng/mL MBP-RavZ protein for 1 h at 37 C.The LC3-negative membranes were achieved by centrifugation at 15,000 rpm, 20 min, 4 C and washed with PBS.

Co-precipitation assay
For co-precipitation assay of protein-palmitate conjugate and total membrane, 20 ng/mL p62 protein or palmitate-maleimide conjugated p62 protein was added into 200 mL total cellular membrane and incubated for indicated time at room temperature, of which 100 mL mixture was transferred to new tube as control (Total).The membrane-binding p62 was separated by centrifugation at 15,000 rpm, 20 min, 4 C, and washed with PBS.The supernatant was transferred to new tube (Supernatant).All samples were resuspended in SDS Sample Buffer and analyzed by Western blotting.
For co-precipitation assay of exogenously expressed Flag-p62 and membrane fraction, the total cellular membrane was resuspended in 100 mL Flag-p62 WT or Flag-p62 C289,290S solution for 1 h at room temperature, of which 50 mL mixture was transferred to new tube as control (Total).The membrane-binding p62 was separated by centrifugation at 15,000 rpm, 20 min, 4 C, and washed with PBS.The supernatant was transferred to new tube.All samples were resuspended in SDS Sample Buffer and analyzed by Western blotting.

Non-reduced SDS-PAGE
The cells were lysed, and dissolved in SDS Sample Buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 2% SDS, 0.1% bromophenol blue) with cocktail inhibitor.To retain the disulfide bonds, reducing agent is not permitted.Samples were boiled at 95 C for 5 min.Oligomer and monomer of p62 were separated by SDS-PAGE (4% stacking gel and 10% separating gel).

Cell death measurement
Freshly trypsinized cells (1 3 10 6 cells) were washed twice with cold PBS and resuspended in 195 mL Binding Buffer (Beyotime, C1065S).Cells were treated with 5 mL propidium iodide (PI, 30 mg/mL) (Beyotime, ST511) and incubated for 15 min at room temperature.After reaction, Cells were transferred to FACS tube, and 800 mL Binding Buffer was added.Then cells were analyzed by the Becton-Dickinson (BD) LSRFortessaÔ flow cytometer.Data was analyzed by FlowJo 7.6.1 software.

QUANTIFICATION AND STATISTICAL ANALYSIS
Unless otherwise indicated, images are representative of cells from at least three separate experiments.Data presented in text and graphs are the means plus standard error of the mean (SEM) and are representative of at least three independent experiments.Unpaired t tests were used to establish the significance of experimentally observed differences.

Figure 3 .
Figure 3. S-acylation facilitates the degradation of p62 and ubiquitinated proteins

Figure 4 .
Figure 4. S-acylation promotes the formation of small LC3-positive p62 puncta (A) Representative images of p62 puncta in GFP-p62 WT or GFP-p62 C289,290S cells.GFP-p62 WT or GFP-p62 C289,290S was stably expressed in p62-KO NRK cells.Cells were fixed with paraformaldehyde and observed under confocal microscopy.The arrows indicate p62 puncta with a diameter of more than 1 mm.Scale bars, 10 mm.(B) Quantification of the number of p62 puncta with a diameter of more than 1 mm in each cell as in (A).***p < 0.001, Student's t test.(C) Immunoblot analysis of p62 proteins in GFP-p62 WT or GFP-p62 C289,290S cells.(D) Representative images of p62 puncta in ZDHHC19-KO HEK293T cells.Scale bars, 10 mm.(E) Quantification of the number of p62 puncta with a diameter of more than 1 mm in each cell as in (D).***p < 0.001, Student's t test.(F) Representative images of p62 puncta in GFP-p62 NRK cells transiently expressing HA-ZDHHC19 WT or HA-ZDHHC19 C142S .Scale bars, 10 mm.(G) Quantification of the number of p62 puncta with a diameter of more than 1 mm in each cell as in (F).***p < 0.001, Student's t test.(H) Representative images of p62 puncta in GFP-p62 WT or GFP-p62 C289,290S NRK cells transiently expressing HA-ZDHHC19.Scale bars, 10 mm.(I) Quantification of the number of p62 puncta with a diameter of more than 1 mm in each cell as in (H).ns, no significant difference.***p < 0.001, Student's t test.See also Figure S4.

Figure 7 .
Figure 7. APT1 mediates deacylation of p62 vation Support Program for Chongqing Overseas Returnees grant cx2022066 to A.Y.; the Fundamental Research Funds for the Central Universities (2022CDJYGRH-002) to A.Y. and T. X.; NSFC and the Royal Society (RS) joint exchange program (22011530161 and IEC\\NSFC\191085) to A.Y. and E.W.T.; and Cancer Research UK (DRCNPG-Nov21\100001, with support from the Engineering & Physical Sciences Research Council) to E.W.T. AUTHOR CONTRIBUTIONS A.Y. conceived the study and supervised the project; A.Y., E.W.T., and X.H. wrote the manuscript; X.H., J.Y., L.L., and J.C. performed most of the experiments.E.K. and J.H. contributed to the construction of ZDHHC19-KO and APT1-KO cells.S.Z. and D.L. contributed to the synthesis of palmitate-maleimide.Q.S. and X.W. contributed to GUV experiments.L.M., C.L., X.C., Y.Y., S.O., F.W., and Z.W. contributed to parts of the experiments.E.W.T., E.K., Q.S., D.N., and T.X.provided insightful suggestions.All the authors discussed the results and commented on the manuscript.DECLARATION OF INTERESTS E.W.T. is a founder and shareholder in Myricx Pharma Ltd. and receives consultancy or research funding from Kura Oncology, Pfizer Ltd., Samsara Therapeutics, Myricx Pharma Ltd., MSD, Exscientia, and Daiichi Sankyo.
(Continued on next page) Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.Polysciences, Inc., 23966-2) was used for transfection of cells according to the manufacturer's instructions.The total DNA plasmid amounts used in this study were in the range of 0.5-2 mg per well in 6-well plates or 10-15 mg per 10 cm dish plated to 80% confluency.WT NRK cells and p62-KO NRK cells were a kind gift from Prof. Li Yu (Tsinghua University).FIP200-KO HeLa cells and FIP200-KO MEF cells were a kind gift from Prof. Min Li (Sun Yat-sen University) and Prof. Yueguang Rong (Huazhong University of Science and Technology), respectively.ATG7-KO HEK293T cells were a kind gift from Prof. Kefeng Lu (Sichuan University).
d Original western blot images and microscopy data have been deposited in the Mendeley dataset and are publicly available upon publication.The DOI is listed in the key resources table.d This paper does not report original code.d